[Analysis of hepatitis C virus proteins in hepatic cells of patients with chronic hepatitis C]. / Analiz belkov gepatita C v kletkakh pecheni bol'nykh khronicheskim gepatitom C.

A correlation between the detection of proteins and an activity of the pathological process was analyzed in a study of the content of the C virus hepatitis (CVH) proteins in hepatic cells of patients with chronic C hepatitis (CCH). The expression of CVH proteins in frozen sections of biopsy samples of 69 CCH patients was evaluated by using the immune-histological method involving original monoclonal antibodies (MCA) to 5 CVH proteins. The results of the detection of proteins in patients were compared with an activity and stage of CCH (by using histological tests and a level of alanine aminotransferase--AAT). A set of the CVH proteins were found in the liver of 74% of patients, i.e. core proteins, NS3, NS4A, NS4B and NS5A--in 28, 43, 43, 55 and 58%, respectively. All studied proteins were detected in the cytoplasm of hepatocytes. Proteins were found in the liver more often as compared with the detection rate of CVH RNA in the blood serum (61%). This demonstrates a high sensitivity of the discussed test at detecting the CVH infection. The accumulation of the core protein was shown to correlate with the presence of the replicative form of CVH RNA in the liver and with a higher level of AAT. The quantity of NS5 A-expressing cells correlated directly with a CCH stage. The quantity of NSB- and NS3-positive hepatocytes correlated negatively with an activity of the inflammatory-and-necrotic processes in the liver. Hyper-fermentation was found more often among the antigen-positive patients. The CCH histological activity was proven to be reliably higher at a simultaneous detection of CCH proteins in the liver and of CVH RNA--in the serum.

Characterization of monoclonal antibodies and epitope mapping of the NS4 protein of hepatitis C virus.

Recombinant DNA containing sequences of HCV NS4 protein was expressed in Escherichia coli cells. Six hybridoma clones producing monoclonal antibodies (MAB) to recombinant NS4 protein (rNS4), aa 1677-1756, were developed. Mapping with a panel of 33 peptides and reciprocal competitive EIA have shown that MAB obtained revealed five antigen determinants, not described earlier: MAB 3F11 and 3F12-one genotype-independent epitope of NS4A (aa 1700-1707) common for genotypes 1, 2 and 3; MAB 1D11-genotype-independent epitope (aa 1713-1728) and MAB 1D3-genotype (subtype 1b)-specific epitope of NS4B (aa 1711-1731); MAB 6B11 and C1-two conformation-dependent determinants in 5-1-1 region. These data indicate that the 5-1-1 region of NS4 protein has a complex antigenic structure and contains at least eight epitopes, including five, revealed in the present work. MAB obtained recognized native viral protein in the cytoplasm of liver cells of patients with chronic hepatitis C. The positive rates of the immunostaining for NS4 antigen using MAB 6B11, 1D11 and 3F12 were 64, 59 and 50%, respectively. It was found that 6B11 MAB to a conformation-dependent epitope much more actively interacts with native NS4 than with the recombinant protein to which MAB was developed. The epitope recognized by 6B11 MAB is highly immunogenic since it induces the B-cell response in all patients investigated with identified anti-NS4 antibodies in blood serum. The MAB panel obtained in this study may become a useful tool for the diagnostic purposes, for the investigation of NS4B function and for the host-viral interactions at the cell level.

[Characterization of a panel of monoclonal antibodies to hepatitis C NS3 recombinant protein ]. / Kharakteristika paneli monoclonal'nykh antitel k rekombinantnomu belku NS3 virusa gepatita C.

Recombinant protein rNS3 imitating helicase region (1356-1459 amino acid residues) of hepatitis C virus (HCV) was expressed in E. coli cells and used for BALB/c mice immunization. Seven hybrydoma clones producing monoclonal antibodies (MAbs) to rHS3 were obtained. All MAbs reacted in ELISA with NS3 protein from Murex anti-HCV Version III and in immunoblotting from RIBA 3. These MAbs detect 5 individual epitopes, 4 of which were conformational and 1 discontinuous. All MAbs could compete for rNS3 binding with serum antibodies from patients with chronic hepatitis C, which suggests that these MAbs can recognize the natural HCV NS3 protein.

[Monoclonal antibodies in the diagnosis of infections caused by the herpes simplex virus]. / Monoklonal'nye antitela v diagnostike infektsii, vyzyvaemykh virusom prostogo gerpesa.

A series of hybridomas producing monoclonal antibodies (McAb), specifically interacting with Herpes simplex virus (HSV) proteins, types 1 and 2, has been obtained. McAb 7c4 and 4f6 have been shown to be highly active in the solid-phase enzyme immunoassay (EIA) and to produce no reaction with HSV antigen in the indirect immunofluorescent assay (IIFA). McAb 2b6, 3e5, 4A, 2C effectively detect McAb in IIFA, but not EIA, while McAb 3d 10 exhibit activity in both biochemical assays. Moreover, as established in this investigation, McAb 4A are active against the protein of HSV capsid, McAb 3d10 and 2b6 detect two individual epitopes on the molecule of ribonucleohydreductase, McAb 2C are specific with respect to surface glycoprotein gB, McAb 7c4 and 416 recognize one or two overlapping epitopes of protein gD. McAb 2C are capable of completely neutralizing the infectious activity of HSV in the in vitro cell system. As determined by IIFA, McAb 4A and 4e5 stain specific inclusions in the nucleus of HSV-infected cells, while McAb 2C stain HSV protein, localized in the cytoplasm. All above-mentioned McAb are active against two common antigenic determinants of HSV 1 and HSV 2. The data obtained in this investigation suggest that the series of McAb under study may serve as the basis for the development of diagnostic test systems for the detection of HSV, types 1 and 2, by EIA and IIFA techniques.

Detection of hepatitis C virus core protein circulating within different virus particle populations.

Progress in studying pathogenesis and increasing the reliability of hepatitis C diagnosis can be achieved by analysis of different forms of virus particles circulating in blood of both patients and infected persons. Detection of hepatitis C virus (HCV) proteins faces two basic difficulties: low concentration of HCV proteins, and their blocking by antibodies. The aim of this work was to develop a method for the detection of nucleocapsid (core) protein in the plasma of HCV-infected persons using monoclonal antibodies (MABs). Twenty-seven anti-HCV-positive donor plasmas were studied of which 21 contained HCV RNA and 6 were negative. The plasmas were centrifuged for 3 hr at 143,000 g and the antigenic activity of core-protein was studied in the pellets by EIA using four MABs able to recognize four nonoverlapping determinants, two at N-terminus and two at C-terminus of recombinant core (1-150 aa). The determinants detected were present in the natural core protein of at least two genotypes (1b and 3a). Maximal efficiency of recombinant protein detection was achieved with 2 MABs, whereas a combination of 4 MABs was necessary for optimal detection of natural core protein. This is indicative of different conformational structures of natural protein and its gene-engineered analog. The sensitivity of core detection by monoclonal sandwich assay was 1 ng/ml in the pellet or 5 pg/ml after normalization to the initial plasma volume. To dissociate immune complexes, the pellet was treated with 2.5 M KBr after first treating the pellet with the nonionic detergent Tween 80 to remove the virus lipid envelope. Using this treatment protocol, core protein was found in 19 of 21 RNA positive plasmas.

[Immunochemical properties and specificity of monoclonal antibodies against N- and C-terminal sites of the recombinant protein of the nucleocapsid of hepatitis C virus (HCcAg)]. / Immunokhimicheskie svoistva i spetsifichnost' monoklonal'nykh antitel v otnoshenii N- i C-kontsevykh uchastkov rekombinantnogo belka nukleokapsida virusa gepatita C (HCcAg).

Highly affine murine monoclonal antibodies (MAB) to recombinant nucleocapsid (core) protein of hepatitis C virus (rHCcAg) expressed in E. coli were obtained. The MABs were analyzed by solid-phase enzyme immunoassay (EIA), immunodot, immunoblotting, and competitive immunochemical analysis. For estimating the epitope specificity of MAB, several immunoreactive fragments of different length were cloned from the HCcAg region overlapping 160 N-terminal amino acid (a. a.) residues. Use of these fragments and the competitive EIA demonstrated that MAB recognize 4 non-overlapping epitopes, 2 of which are localized in the 1-80 a. a. and 2 other in the 80-150 a. a. regions. A protocol of EIA for detecting HCcAg using MABs to two nonoverlapping HCcAg epitopes has been designed. The sensitivity of double-site sandwich is 1 ng/ml for the recombinant protein.

[The protective activity of preparations made from the tick-borne encephalitis virus grown using different cell cultures]. / Izuchenie protektivnoi aktivnosti preparatov iz virusa kleshchevogo éntsefalita, vyrashchennogo s ispol'zovaniem razlichnykh kletochnykh kul'tur.

The preparations of tick-borne encephalitis (TBE) virus grown in swine embryo kidney cell culture have been shown to possess pronounced protective activity per unit of virion protein E in comparison with TBE virus preparations derived from cell culture 4647 and chick embryo cell culture. The antigenic activity of all virus preparations under study has proved to be practically the same. The role of post-translation modifications of TBE virus protein E in the manifestation of some of its biological properties is discussed.

pH-dependent fusion of tick-borne encephalitis virus with artificial membranes.

pH-dependent fusion of TBE virus with artificial membranes was effective at slightly acidic pH with maximum at 6.4. The influence of various changes in E protein conformation on fusion process was studied.

[Measles cellular immunity in patients with multiple sclerosis]. / Korevoi kletochnyi immunitet u bolnykh rasseiannym sklerozom.

The micromethod of leukocyte migration inhibition test was used to study cellular immunity in patients with multiple sclerosis (MS) to measles virus antigens and some other infectious (vaccinia virus, tuberculin) and noninfectious (brain white matter extract) antigens. In MS patients the reaction to measles antigen was weaker than in the control group, while the reactivity to the brain white matter extract was increased. As for the responses to vaccinia virus and tuberculin, these two grous did not differ statistically. The population of MS patients under study was not homogeneous in the intensity of measles cellular immunity (MCI). In the early stages of multiple sclerosis, MCI indices did not differ from those in the control group. MCI was the weakest in those subjects developing MS early in life who showed rapid worsening of the clinical status. Besides, MCI values were age-related: in older age groups they were low both in MS patients and in control subjects.